CN109576189B - Composite microbial inoculum for black and odorous water body and preparation method and application thereof - Google Patents

Abstract

The invention discloses a composite microbial inoculum for black and odorous water and a preparation method and application thereof, wherein the composite microbial inoculum is obtained by separately culturing, mixing, inoculating and fermenting strains including pseudomonas putida, pseudomonas alcaligenes, geobacillus stearothermophilus, paracoccus denitrificans and micrococcus muralis. The bacterial strain metabolic function contained in the composite microbial inoculum has strong synergistic complementarity, the bacterial reproduction speed is high, the production cost is low, the environment is protected, no pollution is caused, the performance is excellent and efficient, the water quality of black and odorous water can be effectively improved, and the COD (chemical oxygen demand) and NH (NH) of the water can be effectively reduced₃N, TP, water odor degree and chroma, and improves water transparency.

Description

Composite microbial inoculum for black and odorous water body and preparation method and application thereof

Technical Field

The invention relates to a composite microbial inoculum for black and odorous water, a preparation method and application thereof, belonging to the technical field of sewage treatment.

Background

Along with the continuous acceleration of the urbanization level in China, the continuous discharge of urban sewage causes eutrophication of water bodies, even blacking and smelling of the water bodies, seriously influences the living environment and life of people and hinders the development of social economy. The water bodies such as rivers, lakes and the like have rich nutrition and black odor, the water bodies are seriously anoxic mainly due to the increase of the content of the nutrient-consuming pollutants in the water bodies, and the organic pollutants are digested by the action of anaerobic microorganisms to generate a large amount of malodorous gases (such as hydrogen sulfide, amine, ammonia, mercaptan and the like) so as to cause the water bodies to smell; metal ions of iron, manganese and the like in water and S^2-Forming metal sulfide to blacken the water body.

At present, the traditional treatment methods of black and odorous water mainly comprise a chemical method (flocculation, adsorption and the like), a physical method (sewage interception, dredging and the like) and a biological method. Wherein, the biological method usually applies microbial preparation to treat black and odorous water, and adds a certain amount of microbial preparation into the black and odorous water to accelerate the degradation of pollutants in the water and enhance the self-cleaning capacity of the water.

In recent years, microbial agents have been applied to treatment of black and odorous water, but still have many problems, such as single microbial effect, instable aging, unsatisfactory treatment effect and the like, which cause difficult wide application. Therefore, research and development of the complex microbial inoculum for treating the black and odorous water body are required to be continuously explored and developed, and research and development of a novel high-efficiency complex microbial inoculum for treating the black and odorous water body become matters which are urgently required to be solved at present.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a composite microbial inoculum for black and odorous water and a preparation method and application thereof. The composite microbial inoculum is mainly used for improving the black and odorous and eutrophication phenomena of water bodies in rivers and lakes, contains bacterial strains with strong synergistic complementarity in metabolic function, has high bacterial reproduction rate, low production cost, environmental protection, no pollution, excellent performance and high efficiency, can effectively improve the water quality of the black and odorous water bodies, and can effectively reduce COD and NH of the water bodies₃N, TP, water odor degree and chroma, and improves water transparency.

The invention relates to a composite microbial inoculum for black and odorous water, which is obtained by singly culturing, mixing, inoculating and fermenting strains including Pseudomonas humilis (CGMCC collection, collection number 1.8931, collection date 2009, 2 months and 10 days), Pseudomonas alcaligenes (CGMCC collection, collection number 1.8050, collection date 2008, 5 months and 8 days), Geobacillus stearothermophilus (ACCC collection, collection number 10253, collection date 2009, 6 months and 13 days), Paracoccus denitrificans (Paracoccus densitificans, CGMCC collection, collection number 1.2907, collection date 2002, 1 month and 18 days) and Micrococcus lyticus (Micrococcus lysolyticus, CGMCC collection, collection number 1.4547, collection date 2005, 6 months and 27 days) and collecting the obtained composite microbial inoculum after independent culture, mixed inoculation and fermentation.

The water content of the composite microbial inoculum is less than or equal to 5 percent, and the viable count is 5 multiplied by 10⁷～1×10⁸CFU/g。

When mixed inoculation is carried out, the volume ratio of the bacteria liquid of the pseudomonas mustards, the pseudomonas alcaligenes, the geobacillus stearothermophilus, the paracoccus denitrificans and the micrococcus muralis is 0.8-1.4: 1.8-2.2: 2.8-3.2: 2.8-3.2: 0.8-1.4, preferably the volume ratio of the bacterial liquid is 1: 2: 3: 3: 1.

the preparation method of the composite microbial inoculum for the black and odorous water body comprises the following steps:

separately culturing Pseudomonas mucida, Pseudomonas alcaligenes, Geobacillus stearothermophilus, Paracoccus denitrificans and Micrococcus muralis; mixing the cultured bacteria liquid, inoculating into fermentation medium (sterilized at high temperature), and fermenting until viable count reaches 5 × 10⁷When the concentration is more than CFU/mL, the thalli are collected through centrifugal filtration and freeze drying, and the composite microbial inoculum is obtained.

The process of separately culturing Pseudomonas mucida, Pseudomonas alcaligenes, Geobacillus stearothermophilus, Paracoccus denitrificans and Micrococcus muralis includes the following steps:

a. respectively selecting four strains of pseudomonas putida, pseudomonas alcaligenes, paracoccus denitrificans and micrococcus muralis, inoculating the four strains into a solid slant culture medium for culture at the temperature of 28 ℃ for 24 hours, inoculating the strains into a liquid culture medium for shaking table oscillation culture after the slant culture medium grows out, wherein the rotation speed of the shaking table is 150 plus 180r/min, the culture temperature is 28-30 ℃, the culture time is 24-36 hours, and when the viable count reaches 1 multiplied by 10, the strains are inoculated into the solid slant culture medium for culture⁸And (3) finishing the preparation of the bacterial liquid in a CFU/mL mode, and respectively obtaining a sludge pseudomonas bacterial liquid, an alkali-producing pseudomonas bacterial liquid, a paracoccus denitrificans bacterial liquid and a wall-dissolving micrococcus bacterial liquid.

In the step a, the solid slant culture medium is prepared by mixing the following raw materials in parts by weight: 10g of peptone, 3g of beef extract, 5g of NaCl, 15g of agar and distilled water are supplemented to 1L, the pH value is 7, and the peptone is sterilized at 121 ℃ for 30min and then cooled for use; the liquid culture medium is prepared by mixing the following raw materials in parts by weight: 10g of peptone, 3g of beef extract and 5g of NaCl, adding distilled water to 1L, adjusting the pH to 7, sterilizing at 121 ℃ for 30min, and cooling for use.

b. Selecting Geobacillus stearothermophilus strains, inoculating the Geobacillus stearothermophilus strains into a solid slant culture medium for culture at the temperature of 37 ℃ for 48 hours, inoculating the Geobacillus stearothermophilus strains into a liquid culture medium for shaking table oscillation culture after the slant culture medium grows out, wherein the rotation speed of the shaking table is 150 plus materials and 180r/min, and the culture temperature is 150 plus materialsCulturing at 35-37 deg.C for 48-72 hr until viable count reaches 1 × 10⁸And (5) completing the preparation of the bacterial liquid in the CFU/mL period to obtain the Geobacillus stearothermophilus bacterial liquid.

In the step b, the solid slant culture medium is prepared by mixing the following raw materials in parts by weight: 5g of glucose, 9g of peptone, 5g of beef extract, 2g of NaCl and K₂HPO₄ 1g、KH₂PO₄0.75g of agar and 15g of agar, adding distilled water to 1L, sterilizing at 121 ℃ for 30min, and cooling; the liquid culture medium is prepared by mixing the following raw materials in parts by weight: 5g of glucose, 9g of peptone, 5g of beef extract, 5g of NaCl and K₂HPO₄ 1g、KH₂PO₄0.75g, distilled water was added to 1L, pH 7, sterilized at 121 ℃ for 30min, and then cooled for use.

When mixed inoculation is carried out, the inoculation volume ratio is 5-15%.

The fermentation medium is prepared by mixing the following raw materials in parts by weight: 15-25g of brown sugar, 9-10g of peptone, 3-5g of beef extract, 2-5g of NaCl and KH₂PO₄ 0.75-1.5g、K₂HPO₄0.8-1g, adding distilled water to 1L, adjusting pH to 7, sterilizing at 121 deg.C for 30min, and cooling.

The temperature of fermentation culture is 30-37 ℃, the fermentation culture time is 3-5d, the stirring speed is 160-200rpm, the ventilation volume is 1:1.5-2, and the stirring interval time is 2 h.

The temperature of freeze drying is-21 ℃, and the freeze drying is carried out until the water content is less than or equal to 5 percent.

The application of the composite microbial inoculum of the invention is to add the composite microbial inoculum into black and odorous water body to accelerate the degradation of pollutants in the water body, enhance the self-purification capability of the water body and improve the black and odorous and eutrophication phenomena of the water body of rivers and lakes. The method specifically comprises the following steps:

step 1: firstly, blocking exogenous solid-liquid waste from entering a polluted black and odorous water body, and preliminarily removing the solid waste in the black and odorous water body in a manual or mechanical salvaging mode;

step 2: detecting the COD content in the polluted black and odorous water body, adding the composite microbial inoculum of the invention into the black and odorous water body according to the COD content, and stocking the waterPlants are grown to recover the water ecological system and the self-purification capacity of the water body. The aquatic plant comprises one or more of herba Eichhorniae, hydrilla verticillata, and herba Swertiae Dilutae, and the sample size is 5 strains/m²。

In the step 2, when the COD content in the black and odorous water body is less than or equal to 100mg/L, the composite microbial inoculum is added according to the proportion of 1/100000 of the water body mass; when the COD content in the black and odorous water body is more than 100mg/L, the composite microbial inoculum is added according to the proportion of 1/10000-5/10000 of the water body mass.

Compared with the prior art, the invention has the beneficial effects that:

1. the geobacillus stearothermophilus can generate enzymes such as protease, amylase, bacteriocin and the like to promote decomposition of organic substances in the water body, effectively inhibit growth and propagation of harmful bacteria in the water body, has the functions of phosphate and potassium dissolution, and can effectively remove the TP content in the black and odorous water body; the P-removing agent can effectively warn to remove N and P while removing COD content by the pseudomonas putida and the pseudomonas alcaligenes; paracoccus denitrificans degradable NH in water body₃The content of N is-N, the odor of the black and odorous water body is eliminated, and the transparency of the water body is gradually improved; the micrococcus muralis can absorb heavy metals, destroy cell structures, provide nutrients for growth and propagation of the first four strains, and promote rapid propagation of the strains.

2. The complex microbial inoculum has strong combination and synergistic capability, is not mutually exclusive, can be mutually coordinated in black and odorous water, can quickly form dominant flora, quickly decompose various nutrient substances in the black and odorous water, degrade harmful substances, eliminate peculiar smell and improve the transparency of the water. The product can be directly put in without complex process, and is convenient for use.

3. The compound microbial inoculum of the invention can achieve the purpose of treating both symptoms and root causes, does not produce secondary pollution, and is safe and efficient.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

Fig. 1 is a diagram illustrating the effect of the complex microbial inoculum provided by embodiment 1 in removing main pollutants in a black and odorous water body in a river.

Fig. 2 is a diagram illustrating the effect of the complex microbial inoculum provided by embodiment 2 of the invention on removing main pollutants from a black and odorous water body in a river.

Fig. 3 is a diagram illustrating the effect of the complex microbial inoculum provided by embodiment 3 of the invention on removing main pollutants from a black and odorous water body in a river.

Detailed Description

The preparation process of the bacterial liquid of each strain used in the embodiment of the invention is as follows:

(1) respectively selecting four strains of pseudomonas putida, pseudomonas alcaligenes, paracoccus denitrificans and micrococcus muralis, inoculating the four strains into a solid slant culture medium for culture at the temperature of 28 ℃ for 24 hours, inoculating the strains into a liquid culture medium for shaking table oscillation culture after the slant culture medium grows out, wherein the rotation speed of the shaking table is 150 plus 180r/min, the culture temperature is 28-30 ℃, the culture time is 24-36 hours, and when the viable count reaches 1 multiplied by 10, the strains are inoculated into the solid slant culture medium for culture⁸And (3) finishing the preparation of the bacterial liquid in a CFU/mL mode, and respectively obtaining a sludge pseudomonas bacterial liquid, an alkali-producing pseudomonas bacterial liquid, a paracoccus denitrificans bacterial liquid and a wall-dissolving micrococcus bacterial liquid.

The solid slant culture medium is prepared by mixing the following raw materials in parts by weight: 10g of peptone, 3g of beef extract, 5g of NaCl, 15g of agar and distilled water are supplemented to 1L, the pH value is 7, and the peptone is sterilized at 121 ℃ for 30min and then cooled for use; the liquid culture medium is prepared by mixing the following raw materials in parts by weight: 10g of peptone, 3g of beef extract and 5g of NaCl, adding distilled water to 1L, adjusting the pH to 7, sterilizing at 121 ℃ for 30min, and cooling for use.

(2) Selecting Geobacillus stearothermophilus strains, inoculating the Geobacillus stearothermophilus strains into a solid slant culture medium for culture at the temperature of 37 ℃ for 48h, inoculating the Geobacillus stearothermophilus strains into a liquid culture medium for shaking table oscillation culture after the slant culture medium grows out, wherein the rotation speed of the shaking table is 150 plus materials for 180r/min, the culture temperature is 35-37 ℃, the culture time is 48-72h, and when the viable count reaches 1 x 10⁸The preparation of the bacterial liquid is finished when the bacterial concentration is CFU/mL,and obtaining Geobacillus stearothermophilus bacterial liquid.

The solid slant culture medium is prepared by mixing the following raw materials in parts by weight: 5g of glucose, 9g of peptone, 5g of beef extract, 2g of NaCl and K₂HPO₄ 1g、KH₂PO₄0.75g of agar and 15g of agar, adding distilled water to 1L, sterilizing at 121 ℃ for 30min, and cooling; the liquid culture medium is prepared by mixing the following raw materials in parts by weight: 5g of glucose, 9g of peptone, 5g of beef extract, 5g of NaCl and K₂HPO₄ 1g、KH₂PO₄0.75g, distilled water was added to 1L, pH 7, sterilized at 121 ℃ for 30min, and then cooled for use.

The following are examples of the preparation and application of the complex microbial inoculum of the invention:

example 1:

bacteria liquid of pseudomonas putida, pseudomonas alcaligenes, geobacillus stearothermophilus, paracoccus denitrificans and micrococcus muralis is mixed according to the volume ratio of 1: 2: 3: 3: 1 mixing, inoculating 10% fermentation medium volume ratio after high temperature sterilization, fermenting and culturing at 30 deg.C for 5 days until viable count reaches 5 × 10⁷When the concentration is more than CFU/mL, carrying out centrifugal filtration and freeze drying at-21 ℃ until the water content is less than or equal to 5 percent, thus obtaining the composite microbial inoculum for treating the black and odorous water body. The fermentation medium is prepared by mixing the following raw materials in parts by weight: 15-25g of brown sugar, 9-10g of peptone, 3-5g of beef extract, 2-5g of NaCl and KH₂PO₄ 0.75-1.5g、K₂HPO₄0.8-1g, adding distilled water to 1L, adjusting pH to 7, sterilizing at 121 deg.C for 30min, and cooling.

The composite microbial inoculum is mainly used for treating black and odorous and eutrophic water bodies of rivers and lakes to reduce COD and NH in the water bodies₃N, TP, eliminating peculiar smell and improving water transparency.

The composite microbial inoculum is adopted to treat the black and odorous river, the side ditch of a certain village is about 500 meters long, the width is about 4-5 meters, the water depth is about 2-3 meters, and the thickness of bottom mud is about 1-1.5 meters. The river channel mainly takes domestic sewage as a main component, a small amount of industrial sewage is gathered, malignant aquatic weeds grow excessively, the phenomena of periodic excessive propagation, putrefaction and excessive propagation are presented, mosquitoes are bred, and foul smell is emitted.

The treatment method comprises the following steps: blocking foreign solid-liquid waste from entering the polluted black and odorous water body, and primarily removing the solid waste in the water body in a manual or mechanical salvaging mode; 1/10000 of the mass ratio of water body is added with the composite microbial inoculum of the invention, and aquatic plants (including water hyacinth, hydrilla verticillata and tape grass, 5 strains/m) are simultaneously bred²) And regulating and monitoring are carried out, so that the water quality index of the river channel is optimized, the biological diversity of an ecological system is increased, and the self-purification capacity of the water ecological system and the water body is restored. And taking a water sample every three days for detection, and observing the water quality change condition. Experimental results show that after the composite microbial inoculum is added, the black and odorous phenomenon is obviously weakened on the 6 th day, the water transparency is improved, the peculiar smell of the water body is obviously weakened, and COD (chemical oxygen demand) and NH (NH) in the water body are obviously weakened after 18 days₃The removal rate of the-N, TP content reaches 92.59%, 88.19% and 93.67%, respectively. The removal effect is shown in figure 1, and the water quality indexes of each stage after treatment are shown in table 1.

TABLE 1 indexes of water quality in different stages of treatment of the black and odorous river

Treatment stage	COD(mg/L)	NH3-N(mg/L)	TP(mg/L)
				Before treatment	416.32	36.78	9.67
After treatment at 18d	30.85	4.34	0.61
				1 month after treatment	29.54	3.21	0.58
After 2 months of treatment	27.86	2.64	0.39
				4 months after treatment	25.32	1.92	0.35
Detection standard	HJ/T 399-2007	HJ 535-2009	GB 11893-89

Example 2:

otherwise, as in example 1, the complex microbial inoculum of the invention is added into water body at 2.5/10000, water sample is taken every three days for detection, and the water quality change condition is observed. Experimental results show that after the composite microbial inoculum is added, the black and odorous phenomenon is obviously weakened on the 6 th day, the water transparency is improved, the peculiar smell of the water body is obviously weakened, and COD (chemical oxygen demand) and NH (NH) in the water body are obviously weakened after 18 days₃The removal rate of the-N, TP content reaches 91.08%, 74.11% and 91.85% respectively. The removal effect is shown in figure 2, and the water quality indexes of each stage after treatment are shown in table 2.

TABLE 2 indexes of the water quality in different stages of the treatment of the black and odorous river

Treatment stage	COD(mg/L)	NH3-N(mg/L)	TP(mg/L)
				Before treatment	421.69	36.48	9.81
After treatment at 18d	37.62	9.44	0.80
				1 month after treatment	35.38	7.85	0.72
After 2 months of treatment	32.48	6.37	0.61
				4 months after treatment	29.87	4.65	0.48
Detection standard	HJ/T 399-2007	HJ 535-2009	GB 11893-89

Example 3:

the other parameters are the same as those of the embodiment 1, the composite microbial inoculum is added into the water 4/10000, water samples are taken every three days for detection, and the water quality change condition is observed. Experimental results show that after the composite microbial inoculum is added, the black and odorous phenomenon is obviously weakened on the 6 th day, the water transparency is improved, the peculiar smell of the water body is obviously weakened, and COD (chemical oxygen demand) and NH (NH) in the water body are obviously weakened after 18 days₃The removal rate of the-N, TP content reaches 91.12%, 72.53% and 91.62% respectively. The removal effect is shown in FIG. 3, and the water quality indexes of each stage after treatment are shown in Table 3.

TABLE 3 indexes of the water quality in different stages of the treatment of the black and odorous river

Treatment stage	COD(mg/L)	NH3-N(mg/L)	TP(mg/L)
				Before treatment	419.87	35.94	9.62
After treatment at 18d	37.28	9.87	0.81
				1 month after treatment	35.84	8.64	0.73
After 2 months of treatment	33.46	6.75	0.64
				4 months after treatment	30.11	5.12	0.52
Detection standard	HJ/T 399-2007	HJ 535-2009	GB 11893-89

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these should be considered as within the scope of the present invention.

Claims (6)

1. The application of the composite microbial inoculum for the black and odorous water body is characterized in that:

the composite microbial inoculum is added into a black and odorous water body to accelerate the degradation of pollutants in the water body, enhance the self-purification capacity of the water body and improve the black and odorous and eutrophication phenomena of the water bodies of rivers and lakes; the method comprises the following steps:

step 1: firstly, blocking exogenous solid-liquid waste from entering a polluted black and odorous water body, and preliminarily removing the solid waste in the black and odorous water body in a manual or mechanical salvaging mode;

step 2: detecting the COD content in the polluted black and odorous water body, adding a compound microbial inoculum into the black and odorous water body according to the COD content, stocking aquatic plants, and recovering the self-cleaning capacity of an aqueous ecosystem and the water body;

in the step 2, when the COD content in the black and odorous water body is less than or equal to 100mg/L, the composite microbial inoculum is added according to the proportion of 1/100000 of the water body mass; when the COD content in the black and odorous water body is more than 100mg/L, the composite microbial inoculum is added according to the mass ratio of 1/10000-5/10000 of the water body;

the compound microbial inoculum is prepared from pseudomonas putida (pseudomonas putida)Pseudomonas borbori) CGMCC 1.8931, Pseudomonas alcaligenes (A)Pseudomonas alcalophila ) CGMCC 1.8050, Geobacillus stearothermophilus (B.stearothermophilus)Geobacillus thermophilus) ACCC 10253, Paracoccus denitrificans: (Paracoccus denitrificans) CGMCC 1.2907 and Micrococcus muralis (C)Micrococcus lysodeikticus) The strain of CGMCC 1.4547 is cultured independently, mixed and inoculated, fermented and then collected to obtain the composite microbial inoculum;

when mixed inoculation is carried out, the volume ratio of the bacteria liquid of the pseudomonas mustards, the pseudomonas alcaligenes, the geobacillus stearothermophilus, the paracoccus denitrificans and the micrococcus muralis is 0.8-1.4: 1.8-2.2: 2.8-3.2: 2.8-3.2: 0.8-1.4.

2. Use according to claim 1, characterized in that:

the water content of the composite microbial inoculum is less than or equal to 5 percent, and the viable count is 5 multiplied by 10⁷~1×10⁸ CFU/g。

3. The application of the composite bacterial agent for black and odorous water bodies as claimed in claim 1, wherein the composite bacterial agent for black and odorous water bodies is prepared by a method comprising the following steps:

separately culturing Pseudomonas mucida, Pseudomonas alcaligenes, Geobacillus stearothermophilus, Paracoccus denitrificans and Micrococcus muralis; obtained after culturingThe obtained bacterial liquid is mixed and inoculated into a fermentation culture medium, and the fermentation culture is carried out until the viable count reaches 5 multiplied by 10⁷ When the concentration is more than CFU/mL, the thalli are collected through centrifugal filtration and freeze drying, and the composite microbial inoculum is obtained.

4. Use according to claim 3, characterized in that:

when mixed inoculation is carried out, the inoculation volume ratio is 5-15%.

5. Use according to claim 3, characterized in that:

the fermentation medium is prepared by mixing the following raw materials in parts by weight: 15-25g of brown sugar, 9-10g of peptone, 3-5g of beef extract, 2-5g of NaCl and KH₂PO₄ 0.75-1.5 g、K₂HPO₄0.8-1g, adding distilled water to 1L, sterilizing at 121 deg.C for 30min, and cooling.

6. Use according to claim 3, characterized in that:

the temperature of fermentation culture is 30-37 ℃, the fermentation culture time is 3-5d, the stirring speed is 160-200rpm, the ventilation volume is 1:1.5-2, and the stirring interval time is 2 h.

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